Our recognition of the fundamental mechanisms mRNA expression, exemplified by the regulated expression of HIV, provided the basics for our interest in developing preventive HIV pDNA based vaccine strategies. Attractive features of the pDNA platform lie in its simplicity, versatility, stability, with repeated administration without vector immunity, being a non-replicating vaccine and not association with adverse effects. Several pDNA vaccines are currently in clinical trials against HIV and cancer and there is a licensed pDNA vaccine against dog melanoma. We are testing immunogenicity of SIV and HIV pDNA vaccines in mice and selected pDNA candidates advance to the macaque model. Some of our successful candidates are now moving to clinical trials. Because intramuscular injection of pDNA induces relatively low immune responses in macaques and humans, we are testing additional delivery methods, including in vivo electroporation and liposomes. We reported that electroporation dramatically increased the efficiency of DNA delivery in naive macaques, leading to greatly augmented antigen expression and resulting in the induction of highest levels of T cell responses. Our pDNA vaccine includes the cytokine IL-12 pDNA (expressed from optimized pDNA) as adjuvant and we reported increased magnitude and quality of the responses. Importantly, we reported the dissemination of the pDNA vaccine induced T cell responses to mucosal sites including rectal and vaginal mucosa, the portal of entry of HIV. We also found that pDNA induced immune responses show extraordinary longevity in vaccinated macaques detectable for several years after the last vaccination. pDNA vaccination elicits moderate humoral immune responses in macaques. We showed that a protein boost can induce higher levels of Ab. We found that co-delivery of pDNA+protein, using either unadjuvanted or adjuvanted protein, in the same muscle at the same time increased Ab production and mucosal dissemination. DNA+Protein co-immunization is superior to vaccination with either of the two individual components in eliciting humoral immune responses. Using pDNA-only vaccination, we found that our optimized DNA vaccine vectors are able to induce potent immune responses able to protect from high viremia. Combination of pDNA+Protein induces potent humoral responses able to significantly delay or prevent virus acquisition and improvement in virological control of the highly pathogenic SIV challenge. HIV Env is a complex highly glycosylated protein. The ever-evolving virus has found uncountable ways to escape the immune system. In addition, the combination of dominant epitopes that distract the immune system are favored over subdominant more conserved epitopes (both for cellular and humoral responses) provide a major stumbling block for the development of a successful vaccine. Since HIV RV144 trial showed modest efficacy associated with responses to the V2 region in Env, we are exploring novel approaches to prime the immune system focusing on V2 epitopes to induce more effective responses. In addition, we are also testing novel protein adjuvants to improve vaccine efficacy. One promising candidate is the combination of TLR4+TLR7 agonist that induced humoral immune responses able to significantly protect against heterologous challenge. In a collaborative effort, we are testing the concept of combining vaccine against rubella and HIV. This regimen includes Gag as immunogen, because Gag-specific T cell responses were reported to correlate with control of viremia in infected individuals. Vaccine regimens including rubella vector and SIV gag DNA in different prime-boost combinations resulted in robust long-lasting cellular responses with significant increase of cellular responses upon boost. In an expansion, a novel HIV gp120 engineered outer domain (eOD) that is targeted by broadly neutralizing antibodies was included in the rubella vaccine. The rubella/eOD vectors stably expressed glycoproteins can bind germline precursors and mature forms of VRC01-class broadly neutralizing antibodies. These vectors potentially could be used as part of a sequential immunization strategy to initiate the production of broadly neutralizing antibodies. Rubella vectors provide a potent platform for inducing HIV-specific immunity that can be combined with DNA in a prime-boost regimen to elicit durable cellular immunity. We continue to build on this vaccine platform for therapeutic approaches. An ideal HIV vaccine should provide protection against all HIV-1 variants. Thus, an important aspect of HIV vaccine development is the selection of immunogens, which has to take into consideration the diversity of the different HIV clades and the identification of the critical epitopes able to induce relevant immune responses, avoiding potential immunodominant decoy epitopes. Indeed, we reported a potent impact of Env on the induction of Gag cellular responses. To address these problems, we are exploring approaches to maximize immunological strength and breadth using mosaic and consensus molecules as well as focusing on highly conserved regions of HIV to induce immune responses to nearly invariable proteome segments, essential for the function of the virus, while excluding responses to variable and potentially immunodominant decoy epitopes. Using the latter, we have developed two distinct approaches focusing on conserved regions of HIV. We developed a prototype vaccine targeting highly conserved regions within the p24gag (p24CE DNA vaccine). Immunogens were engineered encoding conserved elements (CE) of HIV-1 selected on the basis of stringent conservation, functional importance, broad HLA-coverage, and association with viral control. In proof-of-concept studies in mice and macaques, we demonstrated that immunization with CE pDNA elicits robust cellular and humoral immune responses against CE, which cannot be achieved by p55gag DNA vaccination. This vaccine induces potent cytotoxic T cells responses targeting the Achilles' heel of the virus and induces robust immune responses to subdominant epitopes. Importantly, we demonstrated that priming with CE pDNA and boosting with p55gag pDNA greatly augment the CE-specific responses and that inclusion of the p24CE+gag pDNA in the boost maximizes both magnitude and breadth. We identified a novel and effective strategy to maximize responses against Gag and provide a novel strategy to shift the immunodominance hierarchy and to induce robust immune responses to subdominant epitopes. We have further expanded the CE concept to HIV Env. As found for HIV gag CE, we reported that the identified CE in HIV env are subdominant and that our novel DNA vaccine regimen by including a CE DNA prime alters the immunhierarchy and enable induction of robust cytotoxic T cell responses recognizing these epitopes. The vaccine will allows us to expand our vaccine trials to include also therapeutic applications in the macaque model. We are translating the novel HIV CE DNA vaccine concept (CE DNA prime-CE+full length gag DNA boost) to the clinic in an HVTN/DAIDS-supported clinical trial (HVTN 119; open August 24, 2017) with the aim to test whether our p24CE pDNA vaccine concept elicits superior breath and magnitude of Gag responses compared to the optimized immunogen comprising the complete p55Gag protein. cGMP preparations of our pDNA molecules have been generated and tested for potency and immunogenicity. This vaccine includes IL-12 pDNA as molecular adjuvant and in vivo electroporation as pDNA delivery method, two vaccine components our research had shown to be of outmost importance to induce potent T cell responses in macaques. HVTN 119 will combine several of milestones (pDNA expression vectors, adjuvants and delivery) we have achieved over many years in vaccine development.